PROJECT 1: ABSTRACT CTL infiltration of tumor microenvironments (TME) predicts prolonged survival of patients with colorectal cancer (CRC). It also differentiates between the small subset of patients (<5%) with microsatellite instability- high [MSI-H] CRC, who show high levels of intratumoral CTLs and respond to PD-1 blockade from the rest of CRC patients who do not respond. Our preliminary data demonstrate that a) TLR3-based adjuvants induce CTL-attracting chemokines selectively in tumor stroma, but not surrounding non-tumor tissues; b) that combination of TLR3 ligands (such as rintatolimod) with IFN? synergistically induce high levels of CTL- attractants uniformly in all tumor lesions; and c) that inclusion of COX2 blockers enhances specificity of CKM in promoting CTL attraction but suppressing Treg attraction. The three-component chemokine-modulatory regimen (CKM rintatolimod, IFN?? celecoxib) enhanced intratumoral CTL accumulation, prolonged survival and synergized with PD1- and PD-L1 blockers in inducing cures (> 150 day survival) in mice with i.p. MC38 tumors, resistant to PD-1 blockade alone. We completed phase I evaluation of systemic (i.v) CKM in patients with liver- metastatic CRC (NCT01545141), observing its very good tolerability and improved ratios of CTL-to-Treg markers in TME (compared to our patients receiving standard care only). We propose to: Aim 1. Evaluate the in-patient immunologic effectiveness of i.v.- administered CKM to promote local CTL accumulation in liver-metastatic CRC lesions in phase IIa trial NCT03403634. Comparing pre- versus post-treatment tumor biopsies of 12 patients with liver-metastatic CRC, we will test if systemic CKM will abrogate the TME heterogeneity and uniformly increase CTL numbers in TMEs, but not in surrounding tissues. Aim 2. Evaluate the immune and antitumor effects of sequential versus cyclic application of CKM and PD1 blockade and the advantage of additional immunization for long-lasting anti-tumor benefit. In preclinical studies, we will test the hypotheses that the CKM-attracted DCs, NK cells and T cells will a) promote local and systemic tumor-specific immunity and b) will amplify the CKM-initiated intratumoral production of CTL attractants in an IFN? and TNF?-dependent mechanism, resulting in sustained conditioning of the TME for continued antitumor activity of PD1 blockade, even in the absence of additional vaccination. Aim 3. Perform a phase I/II trial to test the clinical activity of CKM combined with PD-1 blockade in patients with microsatellite-stable (MSS) CRC. In phase I/II trial, we will evaluate the clinical efficacy (iORR; iRESIST) of CKM/anti-PD-1treatment in 19 patients with MSS-CRC, traditionally resistant to immunotherapy. Programmatic Role: The unique role of Project 1 is to evaluate the effectiveness, uniformity and tumor- selectivity of systemically-applied CKM and develop CKM-based treatments with sustained anticancer effect. Its success will provide us with a tool to extend the therapeutic benefit of immunotherapy to a particularly large group of patients with MSS-CRC and other cancers currently non-responsive to checkpoint blockade.